Three-dimensional image formation in confocal microscopy

Three-dimensional (3-D) imaging in confocal microscopes is considered in terms of 3-D transfer functions. This leads to an explanation of axial imaging properties. The axial response was observed in both object-scanning and beam-scanning microscopes and the influence of off-axis examination investigated. By simple processing of multi-detector signals, imaging in both the axial and transverse directions can be improved.     

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.     

Data-driven, simultaneous blur and image restoration in 3-D fluorescence microscopy

A novel algorithm for simultaneous blur and image restoration (SBIR)* in three-dimensional (3-D) fluorescence microscopy is presented. All the internal parameters including the point spread function essential for the restoration are estimated from the data. Validation of the SBIR algorithm using simulated signals/images and known real world specimens is provided. Both lateral and axial resolution of images are improved by the application of the algorithm. Finally, the results of the application of the algorithm to unknown specimens are shown, demonstrating the potential of the algorithm in practical applications. Furthermore, evidence is provided to show that this algorithm can provide a turn-key system to deblur images in 3-D fluorescence microscopy.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.     

Fluorescence saturation in confocal microscopy

The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.     

Quantitative fluorescence in confocal microscopy

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen-stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations. In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re-absorption of the excitation light and emission light respectively. In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens. Under confocal observation, as measured with the BIO-RAD MRC-500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.     

基于梯度图的快速POCS超分辨率复原算法研究

随着红外成像相关产业的兴起,红外成像技术具有的隐蔽性好、探测范围广、定位精度高、穿透距离远,以及轻质小巧、低耗可靠等优点备受青睐,已成为当前智能化光电探测发展的主流方向。然而,红外弱小目标的图像细节特征少、信噪比低等特点成为红外图像应用的瓶颈,如何提高红外弱小目标成像效果成为目前的研究热点。POCS算法是目前超分辨率复原中应用非常广泛的一种复原算法,但是该算法运算量大,处理时间较长,同时对图像的边缘细节保留能力较差。针对POCS超分辨率复原算法迭代时间较长,无法满足光电探测系统实时性的问题,提出了基于梯度图的快速POCS超分辨率复原算法(GPOCS)。该算法根据图像的梯度分布对图像中的像素点进行分类,采用不同的迭代系数进行计算。改进算法能够较好的保留边缘信息并抑制噪声,进而在保证超分辨率复原性能的基础上大大缩短了运算时间。实验结果表明:GPOCS算法复原结果在背景处噪声得到一定的抑制,整体复原能力优于传统的POCS复原方法。该算法能够有效地保留边缘细节,同时处理时间小于传统的POCS复原方法,减少了1个数量级已经是接近实时。GPOCS算法能够自适应的选取迭代步长,较好的保留边缘信息并抑制噪声,进而在保证超分辨率复原性能的基础上大大缩短了运算时间,虽然不能满足实时性的要求,但是也已经是接近实时。     

Absorption and scattering correction in fluorescence confocal microscopy

In three-dimensional (3-D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space-dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non-uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3-D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.     

Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy

For deconvolution applications in three-dimensional microscopy we derived and implemented a generic, accelerated maximum likelihood image restoration algorithm. A conjugate gradient iteration scheme was used considering either Gaussian or Poisson noise models. Poisson models are better suited to low intensity fluorescent image data; typically, they show smaller restoration errors and smoother results. For the regularization, we modified the standard Tikhonov method. However, the generic design of the algorithm allows for more regularization approaches. The Hessian matrix of the restoration functional was used to determine the step size. We compared restoration error and convergence behaviour between the classical line-search and the Hessian matrix method. Under typical working conditions, the restoration error did not increase over that of the line-search and the speed of convergence did not significantly decrease allowing for a twofold increase in processing speed. To determine the regularization parameter, we modified the generalized cross-validation method. Tests that were done on both simulated and experimental fluorescence wide-field data show reliable results.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

Automated tracing and volume measurements of neurons from 3-D confocal fluorescence microscopy data

Three-dimensional (3-D) image analysis algorithms and experimental results that demonstrate the feasibility of fully automated tracing of neurons from fluorescence confocal microscopy data are presented. The input to the automated analysis is a set of successive optical slices that have been acquired using a confocal scanning laser microscope. The output of the system is a labelled graph representation of the neuronal topology that is spatially aligned with the 3-D image data. A variety of topological and metric analyses can be carried out using this representation. For instance, precise measurements of volumes, lengths, diameters and tortuosities can be made over specific portions of the neuron that are specified in terms of the graph representation. The effectiveness of the method is demonstrated for a set of sample fields featuring selectively stained neurons. Additional work will be needed to refine the method for unsupervised use with complex data involving multiple intertwined neurons and extremely fine dendritic structures.     

Measurement and restoration of the point spread function of fluorescence confocal microscopy

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero‐centred point spread function.     

Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.     

Three-dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.     

Three-dimensional imaging in confocal imaging systems with finite sized detectors

We consider the effect of the finite size of the detector on both the lateral and axial resolution of the confocal system. The use of a finite sized detector means that the imaging is no longer truly coherent. We find that the lateral resolution is considerably more sensitive to the detector size than is the axial response. The question of the rejection of flare light is also considered. Experimental results are shown and we find that acceptable extended-focus, auto-focus and height images may be obtained from non truly-confocal systems. We also find that lens apodization has a far greater effect on the axial resolution than the lateral resolution.